Cell lines are at the backbone of scientific research and have enabled countless discoveries and preclinical studies. Although there are many cell lines available, researchers have many reasons to generate or modify their cell lines—perhaps to study a particular mutation in a cancer cell line or to determine the effects of knocking down a gene of interest.

To generate a stable cell line, there are three general steps:

Though there are different methods of stable cell line generation, each protocol follows these general steps. The most common methods of stable cell line generation include:

  • Transfection
  • Transduction

We will cover the specifics of each of these methods, as well as the advantages and disadvantages of each method in this article. When generating a cell line, it is important to consider if you require transient or stable expression of your gene of interest. Transient expression of a gene occurs when exogenous DNA introduced into a cell line is only temporarily expressed and does not integrate into the genome [1]. Stable expression of a gene means long-term, continued expression of a gene of interest [1]. Transient expression can be a better method when studying short-term effects in cells since this is a much faster method. However, if cells will be used over multiple experiments or studied for long-term changes, then stable expression is preferred [1].

Which cell type should I use?

Before generating a cell line, it is important to decide which type of cell is needed. First, let’s review the types of cells often used in research.

Primary cells

Primary cells are collected directly from patients (such as their blood, tumor, etc.), processed, and cultured. Compared to other cell types, primary cells are more heterogeneous and offer better biological relevance since they are derived directly from patients, though these cells cannot replicate as much and therefore cannot be cultured as long as other cell types [2].

Immortalized cells

Immortalized cells can grow indefinitely in culture [2]. Cells can be immortalized through the introduction of an immortalization gene (such as SV-40 large T antigen, hTERT, etc.), spontaneous immortalization through passaging, or due to an acquired ability to replicate indefinitely as seen in cancer cells [3-6]. The benefit of using an immortalized cell line is that they are much easier to work with in culture and have a much longer ‘lifespan’ as they can be cultured nearly indefinitely. However, immortalized cells often have less biological relevance compared to primary cells and may have additional genetic mutations [2].

Stem cells

Stem cells are unique in their ability to differentiate into multiple cell types and self-renew [7]. Stem cells are classified based on their differentiation capabilities: totipotent stem cells can differentiate into any cell type including placental cells; pluripotent stem cells can differentiate into any cell type; multipotent stem cells can differentiate into certain cell types but not as many as pluripotent stem cells; oligopotent stem cells can differentiate into fewer, more specialized cell types; and unipotent stem cells can differentiate into only one cell type [7].

Cancer cell line

Cancer cell lines are derived from a tumor and generally maintain the specific attributes of that particular cancer [8]. Since these cells are taken and cultured from cancer, they have the ability to replicate indefinitely due to pre-existing mutations [8]. Starting with the first cancer cell line generated (HeLa cells), cancer cell lines have been instrumental in cancer research to help understand the basic biology of the disease and to screen for potential cancer treatments [8].

Methods of Generating a Stable Cell Line:

Transfection

Transfection is a process in which exogenous DNA or other nucleic acids are taken up into eukaryotic cells [9]. Typically, plasmids containing a gene of interest and a selectable marker (ex. Hygromycin, puromycin) or a reporter gene (GFP, RFP) will be transfected into a cell line of interest. The gene of interest will only be transiently expressed in transfected cells unless cells are put into selection typically 48-72 hours post-transfection. Selection with antibiotics, like puromycin, will ensure that only cells successfully expressing the gene of interest can survive. The dosage of selection will depend on the cell line, so it is recommended to do a dose curve before transfection to determine the best concentration needed to eliminate non-transfected cells. With stable cell line generation, a small percentage of transfected cells can incorporate the exogenous DNA into the genome [9].

The general protocol for transfecting cells to create a stable cell line is simple. Cells are transfected with a plasmid (containing a gene of interest and a selectable marker) via chemical-based transfection (lipofection) or non-chemical-based transfection (electroporation) [9]. After recovery for 48-72 hours, transfected cells are put into selection for several days to a few weeks. Once cells have been selected and begin to expand, there are two options: 1) continue experiments with a pool of transfected cells, or 2) isolate single cells and expand to work on a homogenous, genetically identical cell line. Though it is assumed that the pool of transfected cells is equal, integration of plasmid DNA into the genome is random and expression of the gene of interest may vary. It is recommended to check for expression of your gene of interest using western blot method and/or RT-qPCR.

Chemical-based transfection and non-chemical-based transfection

One of the most common chemical-based transfection methods is lipofection. Lipofection relies on the formation of positively charged lipid-DNA complexes that can pass through the phospholipid bilayer of cells, delivering the plasmid DNA into the cells [9,10]. Though there is still much unknown about the specifics of this process, lipofection works well for many cell lines.

Electroporation is a non-chemical-based transfection method that relies on electricity to shock the cells and temporarily cause openings in the cell membrane, allowing for plasmid DNA to enter [9]. This method is known to be harsher on cells and leads to higher cell death, though it is more effective in cells resistant to transfection, like primary cells [9].

Transduction

Transduction is a process in which exogenous nucleic acids are delivered into eukaryotic cells by viral vectors [9]. The viral vectors used in transduction are replication-defective, meaning they cannot replicate on their own without assistance from packaging cell lines and helper plasmids that provide the necessary genes for viral packaging [11]. Once the virus is produced and collected, it can be used to transduce a cell line of your choice. The benefit of adding exogenous DNA to a cell line via viral methods is that most viral vectors produce stable cell lines by integrating the DNA into the host genome [11]. Additionally, difficult-to-transfect cell lines may have a much higher transduction efficiency as opposed to typical transfection methods [11]. Thus, several viral vectors can be employed in experiments to generate a stable cell line. Here we will briefly discuss some of these viral vectors and the advantages and disadvantages of each.

Retrovirus

Retroviruses are single-stranded RNA (ssRNA) viruses that reverse transcribe their RNA into double-stranded DNA (dsDNA) once inside the host cell [11]. The dsDNA is then stably integrated into the host genome, providing an efficient way to generate a stable cell line with your gene of interest [11]. Retroviruses can transduce most mammalian cells, though their ability depends on how the virus was packaged. For example, retroviruses packaged in an ecotropic packaging cell line will be able to infect murine cells, while retroviruses packaged in an amphotropic cell line can infect human, murine, and rat cell lines [12]. It is important to keep in mind how you will package your retrovirus based on which cell line (human, murine, etc.) you would like to generate.

Lentivirus

Lentiviruses are part of the retroviral family, specifically, lentiviruses are complex retroviruses [11]. Similar to retroviruses, lentiviruses are ssRNA viruses that reverse transcribe their ssRNA into dsDNA in the host cell, which is subsequently integrated into the host genome [11]. Interestingly, lentiviruses can infect non-dividing and terminally differentiated cells, which may aid in the generation of a stable cell line that is already terminally differentiated [11]. Lentiviruses can be produced using the lentiviral vector and helper plasmids in either 293T cells or packaging cell lines for greater lentiviral production efficiency [12].

Method Advantages Disadvantages
Transfection
  • Safer
  • Simpler procedure
  • Low transfection efficiency in certain cell lines
Transduction
  • Higher efficiency
  • Good with difficult-to-transfect cell lines
  • Retroviral and lentiviral vectors stably integrate into genome
  • May disrupt other genes when integrating into genome

Types of Stable Cell Lines:

Now that we have covered transfection and transduction, we will discuss common ways these methods are used to create different types of stable cell lines. The most common types of stable cell lines are:

  • Overexpression cell lines
  • Reporter cell lines
  • shRNA knockdown cell lines
  • CRISPR-edited cell lines

What type of stable cell line you choose to generate depends on the goals of your experiments. Overexpression cell lines are relatively simple and fast to produce if you need to overexpress your gene of interest in the cell line of your choice. Reporter cell line generation is ideal if you need to visualize or track your protein of interest in cells. shRNA knockdown cell lines are a great choice if you need to decrease levels of your protein of interest, but do not want to remove expression entirely. Finally, CRISPR-edited cell lines can be time-consuming and labor-intensive to create but can be more biologically relevant since you can mutate or knock out an endogenous gene of interest. For more information on each of these types of stable cell lines and a brief outline of how to generate them, continue reading below.

Overexpression Cell Lines

Overexpression cell lines are typically the simplest to produce in the lab. To generate an overexpression cell line, a plasmid containing your gene of interest and a selectable marker (such as puromycin or hygromycin) is transfected into a cell line of your choice. Expression of the gene of interest can be transient (temporary) or stable (long-term) if cells are put into selection. Generally, expression of the gene of interest can be detected 48-72 hours post-transfection, at which point it is recommended to collect your transfected cells and confirm expression through western blot (to determine protein levels) or through RT-qPCR (to determine mRNA levels). Tagging your gene of interest with an epitope tag can help with specific detection of your gene so that endogenous levels of your gene of interest do not interfere with detection. As mentioned previously, cells can be transfected through a variety of methods, but common methods include chemical-based (lipofection) and non-chemical-based (electroporation) transfection.

Reporter Cell Lines

Reporter cell lines can be produced in many ways (overexpression plasmid, viral vector, gene knock-in), but they all allow for a transcription factor or protein of interest to be monitored, tracked, or visualized in a cell [13]. Generally, your gene of interest will be tagged with fluorescent reporters such as GFP, RFP, or any tag that provides easy visualization or tracking.

shRNA Knockdown Cell Lines

shRNA (short-hairpin RNA) knockdown cell lines allow for decreased expression of a target gene in a cell line of your choice. shRNA can be either transfected (usually lipofection) or transduced using viral vectors into cells [14]. Once present in cells, shRNA is transcribed and processed, turning into mature shRNA. Mature shRNA can then promote the cleavage of a target gene of interest, therefore knocking down expression [14]. The use of shRNA often is much simpler than knocking out a gene via CRISPR/Cas9 editing, though it is recommended to test out several shRNAs and quantify the decrease in expression of the target gene before conducting experiments.

CRISPR-edited Cell Lines

The CRISPR (Clustered Regularly-Interspaced Short Palindromic Repeats)/Cas9 revolution as a method of gene editing over the past decade has certainly changed the way researchers generate cell lines [15]. Most often, CRISPR is used to knock-out or delete a gene, knock-in a gene or epitope tag, or generate a point mutation in a gene of interest with great precision and accuracy. It is possible to create a homozygous or heterozygous insertion or deletion in your cell line, depending on what is most relevant to your experiment. The main components of CRISPR/Cas9 include tracrRNA (trans-activating crRNA), crRNA (CRISPR RNAs), and the Cas9 nuclease [15]. When bound together, the tracrRNA and the crRNA are referred to usually as the sgRNA (single guide RNA). The tracrRNA provides a scaffold for Cas9 to bind, the crRNA provides a unique guide for targeting your gene of interest, and the Cas9 does the ‘cutting’ of DNA [15].

Generating a knock-out cell line

Once DNA is cleaved, there are two ways in which it can be repaired: non-homology end joining (NHEJ) or homology-directed repair (HDR) [15]. NHEJ is the ‘messier’ repair system as it fuses the cleaved DNA back together, often with large deletions that can disrupt a gene and silence its expression [15]. This is a great method if the goal of your experiment is to generate a knock-out cell line with CRISPR.

Generating a knock-in cell line

If your goal is to generate a point mutation or insert a segment into a gene of interest, then you require HDR as the method of DNA repair. HDR requires an HDR oligo donor template that contains the insertion or point mutation you wish to generate in your cell line [16]. Notably, HDR occurs at a much lower rate than NHEJ, so the efficiency of generating the precise insertion or point mutation tends to be low [16]. After utilizing the CRISPR/Cas9 and HDR oligo donor template in your cell line, the laborious task of screening single-cell clones remains. It is important to screen your cells, whether you are creating a knock-out, knock-in, or point mutation in your cells with CRISPR, to ensure that unintended deletions or additions were not added to your gene of interest. While generating a CRISPR-edited knock-in cell line can be much more difficult than other methods of creating a cell line, the reward is a biologically relevant, stable cell line that can be used for countless experiments.

Summary

Stable cell line generation presents unique challenges. Every cell line is different in terms of its transfection or transduction efficiency, so it is common to troubleshoot initially to find the best method of introducing your gene of interest. Many common cell lines, such as HEK293T and 3T3 cells, have well-defined protocols online available for generating a stable cell line. Primary cells are notorious for being difficult to transfect, hence more troubleshooting may be necessary. Non-chemical-based transfection, such as electroporation, is ‘harsher’ but can increase transfection efficiency in certain cell lines. Viral transduction also boasts greater efficiency in several cell lines. When using CRISPR/Cas9 systems to create a stable cell line, it may help to test different sgRNAs to find the one with the best editing efficiency for your gene. Many new nucleases for use in CRISPR editing have been discovered, so it may be beneficial to research which nuclease would be best for your target gene. Overall, there are several methods to generate a stable cell line and a variety of considerations to determine which way is best for your experiments.

Frequently Asked Questions

  • What is stable cell line generation?

    Stable cell line generation involves introducing an exogenous gene of interest into a cell line for long-term expression of the gene of interest. Stable cell line generation can be as simple as transfecting a cell line with an overexpression plasmid or as intricate as using CRISPR/Cas9 for cell line generation.

  • How to generate stable cell lines?

    There are several ways to generate a stable cell line. One method is to transfect cells with an overexpression plasmid and select positively transfected cells. Another method involves transducing cells with a lentivirus or retrovirus to have stable integration of a gene of interest. Another permanent method for stable cell line generation involves CRISPR/Cas9 to knock-in a gene of interest, knock-out or delete a gene of interest, or even generate a point mutation in a gene of interest.

  • Are lentivirus or retroviruses better for stable cell line generation?

    The shortest answer to this question is: it depends. Retroviruses have excellent transduction efficiency and can transduce human, murine, and rat cell lines. However, lentiviruses can transduce terminally differentiated cells and non-dividing cells, so using a lentivirus would be better if your cell line fits these characteristics.

  • Can 293T be used to generate a lentivirus cell line?

    Yes, 293T cells can be used to generate a lentivirus cell line. You will need to add your lentiviral vector and helper plasmids with necessary viral genes to 293T cells to produce a properly packaged lentivirus.

  • How long does it take to generate a cell line?

    It can take anywhere from a few days to several months to generate a stable cell line, depending on the method used. Transfecting cells with an overexpression plasmid is a relatively quick procedure, as cells are often expressing the gene of interest around 48-72 hours post-transfection. However, it could take several weeks or months to isolate a properly edited single-cell clone when using CRISPR/Cas9 to generate a stable knock-in cell line.

  • How to generate a knockout cell line? How to generate genomic deletions in mammalian cell lines via CRISPR/Cas9?

    If your goal is to knock-out a gene of interest, CRISPR/Cas9 is an excellent system. By using a sgRNA to target your gene of interest, a Cas9 nuclease will cleave the gene and non-homology end joining (NHEJ) will ‘glue’ the DNA back together, often with deletions that render the gene silenced.

  • How to generate a mutant cell line?

    It is possible to generate a mutant cell line using an overexpression plasmid with your gene of interest (with an oncogene, mutant tumor suppressor gene, etc.). Alternatively, you could use a viral vector (lentivirus, retrovirus) to introduce a mutant gene that would be stably integrated into the genome. As an additional method, you could use CRISPR/Cas9 to induce a point mutation in your cell line.

References

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